Angiopoietin-mediated NETosis is dependent on ROS production. Neutrophils were stimulated with angiopoietins for 3 h and stained with a cellular ROS-sensitive fluorogenic dye (DCFDA, 20 μM, 30 min), and when applicable, neutrophils were pretreated for 30 min with control vehicle (DMSO 0.1%) or with an NADPH oxidase inhibitor (DPI, 25 μM) (A and B). NETs were isolated and quantified as dsDNA (nanogram per milliliter) by fluorometric assay (Quant-IT PicoGreen dsDNA Assay Kit) (C). Data are mean ± SEM from at least six independent experiments. Each experiment employed neutrophils isolated from a different donor. Dotted line corresponds to control basal level. Significance of data (one-way ANOVA) is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with control PBS, by †p < 0.05, ††p < 0.01, and †††p < 0.001 as compared with control DMSO, and by §p < 0.05 and §§§p < 0.001 as compared with corresponding angiopoietins plus DMSO treatment.